Tick-borne diseases are increasingly being diagnosed in humans and animals. Some are due to the resurgence of previously known illnesses, like Rocky Mountain spotted fever, but others are due to new, emerging pathogens. Among the spotted fever-group (SFG) alone, 8 new human pathogens have been described in the last 15 years, but they also include viruses, ehrlichias and Babesia. Novel, efficient, specific and environmentally acceptable methods that interfere with disease transmission by ticks are urgently needed. Using paratransgenic ticks that carry specific symbiotic prokaryotes expressing an antimicrobial substance, as has been achieved with the symbiote of the Chagas disease vector, Rhodnfus prollxus, could offer a safe and effective way to reduce disease transmission by ticks. A major obstacle to accomplishing this goal has been the lack of culture systems for tick symbiotes. We have in our laboratory the largest collection of tick cell lines: We have successfully used these to isolate tick symbiotic rickettsiae from the Lone Star tick (Isolate MOAa) and the Rocky Mountain wood tick (R. peacockii isolate -DAE100R}. We have characterized these microbes by light and electron microscopy by using specific -antibodies, as well as by PCR and nucleotide sequence analysis of 16S rDNA and other key genes. We are now in the process of defining the cultures to facilitate genetic manipulation of the symbiotes. Our long term aim is the stable transformation of Rickettsia Peacockii with the cecropinA an insect porin gene, infection of ticks with the transformed rickettsia, and interference with pathogen transmission. We plan to target the non-functional rompA gene of R. peacockii as a site for homologous transformation, avoiding deleterious effects associated with disruption of a vital gene, e.g., the rpoB gene. We will take the recent advances in the successful transformation of insect-borne rickettsiae as a guide specifically we will 1): optimize culture conditions for production of R. peacockii in tick cell culture, verify identity of the isolate as R. peacockii by molecular analysis, and examine its behavior in mammalian and tick cell culture by light and electron microscopy.-2) We will characterize and analyze R. peacockii in vivo in ticks in terms of tissue tropisms and transstadial/transovarial passage. Finally, we will work towards 3. stable transformation of A. peacockii with cecropina will then test the transformants for antimicrobial activity in vitro and in ticks, and by sequence analysis.